Biological investigation of Paget cells using immortalized Paget cells

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We have obtained cultured Paget cell which showed an extended life span derived from the involved skin of a patient with extramammary Paget's disease. In one culture grown in low calcium media, a colony continued to proliferate and nearly covered the dish. After 40th subculture, the cells became larger in size, and ceased proliferating at 48th subculture. Population doubling time was -50 hours. As tumorigenecity test, tuumor formation was assayed using SCID mice, but, any tumor formation was not observed. Every cell expresses EMA, however, a few cells express carcinoembryonic antigen, and are positive for PAS staining. When cultured in high CィイD1++ィエD1 medium, cells express desmoglein 1 and 3, and stratified. Kratin analysis was done with two-dimensional gel electrophoresis. Keratin subunits K1, 4, 5, 7, 8, 10, 18 were observed. The amount of K14 was small. The chromosomal constitution of immortalized Paget cells was analyzed, and distinct numerical and structural karyotypic alterations were observed. Genomic DNA was isolated from 38th-passage cultured cells, and exons 5, 6, 7, 8 of p53 genes were amplified by polymerase chain reaction. A missense mutation at codon 248 (CGG to CAG) resulting in a Arg to Gln substitution was identified in exon 7, and this may be a reason why the cells could be maintained longer. A p53 mutation alone, however, does not make a cell immortal. Other mutations of as yet undetermined genes in addition to p53 gene are deemed to be necessary for immortallization of human cells. At present, no isolated Paget cell line for biochemical and molecular investigations is available. The cells we obtained might serve as a promising tool for studying the biology of Paget cells, even though they do not represent a cell line.